New & Noteworthy

When Polymerases Collide

October 25, 2012

Lots of recent studies are showing that transcription happens over way more DNA than anyone previously thought. For example, the ENCODE project has shown that most of a genome gets transcribed into RNA in humans, fruit flies and nematodes. This transcriptional exuberance was recently confirmed in the yeast S. cerevisiae as well.

There is also a whole lot of antisense transcription going on. Taken together, these two observations suggest that there are lots of opportunities for two polymerases to run headlong into each other. And this could be a big problem if polymerases can’t easily get past one another.

Goats butting heads

What happens when RNA polymerases meet head-on?

Imagine that the two polymerases clash in the middle of some essential gene. If they can’t somehow resolve this situation, the gene would effectively be shut off. Bye bye cell!

Of course this is all theoretical at this point. After all, smaller polymerases like those from T3 and T4 bacteriophages manage to sneak past one another. It looks like this isn’t the case for RNA polymerase II (RNAPII), though.

As a new study by Hobson and coworkers in Molecular Cell shows, when two yeast RNAPII molecules meet in a head on collision on the same piece of DNA, they have real trouble getting past each other. This is true both in vitro and in vivo.

For the in vivo experiments, the authors created a situation where they could easily monitor the amount of transcription close in and far away from a promoter in yeast. Basically they pointed two inducible promoters, from the GAL10 and GAL7 genes, at one another and eliminated any transcription terminators between them. They also included G-less cassettes (regions encoding guanine-free RNA) at different positions relative to the GAL10 promoter, so that they could use RNAse T1 (which cleaves RNA at G residues) to look at how much transcription starts out and how much makes it to the end.

When they just turned on the GAL10 promoter, they saw equal amounts of transcription from both the beginning and the end of the GAL10 transcript. But when they turned on both GAL10 and GAL7, they saw only 21% of the more distant G-less cassette compared to the one closer to the GAL10 promoter.

They interpret this result as meaning the two polymerases have run into each other and stalled between the two promoters. And their in vitro data backs this up.

Using purified elongation complexes, they showed that when two polymerases charge at each other on the same template, transcripts of intermediate length are generated. They again interpret this as the polymerases stopping dead in their tracks once they run into one another. Consistent with this, they showed that these stalled polymerases are rock stable using agarose gel electrophoresis.

Left unchecked, polymerases that can’t figure out how to get past one another would obviously be bad for a cell. Even if it were a relatively rare occurrence, eventually two polymerases would clash somewhere important, with the end result being a dead cell. So how do cells get around this thorny problem?

King Arthur and the Black Knight

To get past the Black Knight, Arthur had to destroy him. Hopefully the cell has more tricks up its sleeve than that!

One way is to get rid of the polymerases. The lab previously showed that if a polymerase is permanently stalled because of some irreparable DNA lesion, the cell ubiquitinates the polymerase and targets it for destruction. In this study they used ubiquitin mutants to show that the same system can work at these paused polymerases too. Ubiquitylation-compromised yeast took longer to clear the polymerases than did their wild type brethren.

The authors think that this isn’t the only mechanism by which polymerases break free though. They are actively seeking factors that can help resolve these crashed polymerases. It will be interesting to see what cool way the cell has devised to resolve this dilemma.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: transcription, RNA polymerase II, ubiquitin-mediated degradation, Saccharomyces cerevisiae

Better Beer Through Beards

October 16, 2012

Small time craft brewers are always looking for ways to push the envelope of beer taste.  They are trying to find variations in beer’s fundamental ingredients — hops, barley, and yeast — that will make their beer distinctive.  Of these three, the most important is probably yeast (of course, we’re biased here at SGD!).

Think of the tasty beers that could come out of those beards.

Something like 40-70% of beer taste comes from the yeast used to make it alcoholic.  This is why brewers search high and low for new strains of yeast that will give their beer that special something which will make it stand out.  They have looked on Delaware peaches, ancient twigs trapped in amber, Egyptian date palms, and in lots and lots of other places. 

But brewers don’t always have to go far away because sometimes the best yeast is right under their noses.  Literally.

A brewery in Oregon found the yeast they were looking for in one of their master brewers’ beards.  They are now using this yeast to brew a new beer!  This seems uniquely revolting but the beer supposedly is quite tasty.  Perhaps if they don’t advertise the source of their yeast, this beer could become popular.

They aren’t sure where the yeast in his beard came from, but they think it may have come from some dessert he ate in the last 25 years or so (he hasn’t shaved his beard since 1978).  What would be fun is if his beard wasn’t just an incubator, but a breeding ground for new yeast.  Maybe yeast from a dessert from 1982 hooked up with a beer yeast blown into his beard while he was working at the brewery.  The end result is a new improved hybrid yeast! 

Of course we won’t have any real idea about this yeast until we get some sequence data from it.  And all kidding aside, the more yeast that are found that are good for making beer, the better the chances that scientists can home in on what attributes make them beer worthy.  So this beard borne yeast may help many beers in the years to come despite its troubling beginning.

Perhaps brewers also need to start searching through more beards to look for likely beer yeast candidates.  Beard microbiome project anyone?

More information

About beard yeast

What’s in a beer, anyway?

Original lager yeast found in Patagonia

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: beer, Saccharomyces cerevisiae, fermentation

SGD Hardware Update

October 09, 2012

We are in the process of migrating SGD servers to new faster hardware! You may have already noticed an increase in performance. There could be some teething issues in the next couple days – so please bear with us!

Categories: Website changes

How To Remember Without a Brain

October 02, 2012

Single celled beasts like the yeast S. cerevisiae can “remember” previous insults and so respond better to environmental changes in the future. For example, a yeast cell treated with 0.7M NaCl will respond better in the future to hydrogen peroxide. Not only that but so will its daughters, granddaughters and even its great, great granddaughters.

Who needs a brain to remember things?

In a new study out in GENETICS, Guan and coworkers show at least a couple of ways that this can happen. One is what anyone in biology might expect these days (although with an interesting twist). The NaCl treatment causes a rewiring of the regulatory network at an epigenetic level and this affects future responses to environmental insults.

But fancy epigenetic changes aren’t the only way that yeast remembers things.  No, it also uses a simple, elegant solution—protein stability.
Long Live The Protein!

The researchers did a set of experiments that showed that a yeast’s memory of a salt treatment did not rely on new protein synthesis and that it slowly faded with each generation.  One possible explanation was that the salt induced a stable factor that was divvied up and diluted with each passing generation. Guan and coworkers found that this was the case and that at least one of these factors was the cytosolic catalase 1 protein, Ctt1p.

The cytosolic catalase 1 or CTT1 gene is induced by salt but quickly returns to normal levels when the salt is removed.  However, Ctt1p is so long lived that it hangs around for at least six hours.  In that time the yeast has budded off multiple daughters, all of which are still better at dealing with hydrogen peroxide than their untreated sisters.

What a marvelously simple way to adapt!  Just make something that hangs around a long time and you and your kids will do better when the next insult comes.  The elegance of evolution.

This explains in part how yeast cells can remember the salt treatment of their ancestors, but a single long-lived protein isn’t the whole story.  No, there is something a bit more complicated going on at the nuclear pore too.

Attached for Quick Access

Guan and coworkers looked at the gene expression pattern of salt stressed and naïve yeast when exposed to hydrogen peroxide.  They found that 449 genes responded more quickly to hydrogen peroxide treatment if the cell had been pretreated with salt.  Importantly, 51 of these hadn’t reacted previously to the salt treatment, meaning that previous activation wasn’t required.

One idea is that these genes are more accessible to transcription because they are associated with the nuclear pore.  The idea is that faster response happens because the gene is closer to the nuclear envelope and/or because it has been looped near some sort of activator.

This is what has been proposed with inositol starvation and it looks like it may be true here too.  In both cases, eliminating Nup42p, a nuclear pore protein, eliminates the more rapid response to hydrogen peroxide.

So in this case it looks like cells can remember a previous insult with just a long-lived protein and a bit of genetic rewiring.  It will be interesting to see how universal these sorts of mechanisms are for cell memory.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: cellular memory, Saccharomyces cerevisiae, catalase, stress response

Wintering in a Wasp’s Gut

September 24, 2012

Anyone reading this blog probably knows how important the yeast S. cerevisiae is.  It makes our bread better, our beer and wine more spirited, and our genetics more understandable. 

Social wasps are a natural reservoir for yeast

Because it is such an important beast, this yeast is also incredibly well characterized.  It was the first non-bacterial organism whose genome was sequenced and is a key model organism for teasing apart how eukaryotes like us work. We may know more about the molecular biology and genetics of S. cerevisiae than about any other organism on the planet.

And yet we know surprisingly little about S. cerevisiae in the wild.  We know that it isn’t on unripe fruits but suddenly appears once they ripen. We also know it doesn’t tolerate winter particularly well.  So where does yeast hang out when there isn’t ripe fruit around and/or it gets chilly?  A group of researchers in Italy thinks a key place is inside a hibernating wasp.

When Stefanini and coworkers looked, they found lots of yeast (including S. cerevisiae) in wasp intestines. They were also able to show that the S. cerevisiae remained viable in a hibernating queen over the winter and that that the queen transferred the yeast to new wasps in the spring by regurgitation.  With this one study, these scientists managed to find at least one way that yeast can survive the winter and get to ripe fruit.

To figure this out, Stefanini and coworkers did experiments both in the field and in the lab.  They first collected wasps and bees from around the Italian countryside and showed that wasps, but not bees, harbored yeast in their gut.  In all they found 393 yeast strains in the 61 wasps they dissected, 17 of which turned out to be S. cerevisiae.   By sequencing and comparing the genes URN1, EXO5, and IRC8, they were able to conclude that these yeast were related to wine, beer, bread, and laboratory strains of S. cerevisiae.

The researchers figured out that the yeast could survive for three months and be passed on to the next generation of wasps with a couple of controlled experiments they did in the lab.  They fed queens GFP labeled yeast and then let them hibernate.  After three months they dissected some of them and found lots of viable yeast in their intestines.

The rest of the queens were allowed to wake up and find new nests.  Larvae were removed from the nests and were found to contain GFP yeast as well.  The yeast not only lived through the winter but passed on to the next generations!

Of course this doesn’t mean that this is the only way that it can happen.  But it is the first time anyone has managed to get such a detailed look at feral yeast.  And this kind of work is important if we want to use S. cerevisiae as a way to study evolution. 

To understand its evolution, we have to understand the natural forces that shaped S. cerevisiae into the organism it now is.  Only then can we piece together why S. cerevisiae has evolved the way that it has and so learn fundamental lessons about the mechanisms of evolution. 

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: evolution, Saccharomyces cerevisiae, wild yeast

Few Genetic Paths From Here to There

September 12, 2012

Everyone knows that when the environment changes, those individuals with certain DNA differences useful in this new environment thrive while others wither.  But there hasn’t been a lot of work done to investigate how many DNA differences are available to a population for adapting to a particular environmental change.

How many paths lead to adaptation?

This may sound esoteric but the answer has real implications for speciation.  If there are few mutations possible and these mutations are very similar in terms of phenotype, then different populations will travel similar routes in their adaptations to the same environmental change.  This will definitely slow down speciation.  If on the other hand there are many genetic ways to adapt to the same change, then isolated populations will head down different paths leading to faster speciation.

In a new study out in GENETICS, Gerstein and coworkers found that at least for the environmental insult they used (low levels of the fungicide nystatin), there were very few paths to resistance. In fact, just four genes in the ergosterol biosynthesis pathway turned up in the 35 resistant lines they surveyed using whole genome sequencing.

Now that isn’t to say that there were just a few mutations.  There weren’t.  They found eleven unique mutations in the ERG3 gene, seven in ERG6, and one each in ERG5 and ERG7.  There were duplications, deletions, premature stop codons and missense mutations.  So there are lots of ways to mutate these few genes.

The small range of genes affected might suggest that adaptation favors populations evolving along similar paths since the same environmental effects result in the same adaptative mutations.  And yet, not all of these mutations in these few genes are created equally.  Different lines responded differently to other stressors.

For example, lines with mutations in the ERG3 gene responded poorly to ethanol while the other lines did very well.  And the lines with mutations in ERG5 and ERG7 responded less well to salt than the other lines.  So if one population was subjected to salt and nystatin and the other to ethanol and nystatin, the strains would almost certainly adapt with mutations in different genes.  Even within this narrow set of genes, there is room for adaptation by different routes.

While a useful first step, we don’t want to infer too much from this single study.  The researchers used a very specific environmental insult known to work through a specific pathway and found only mutations in that pathway.  The next study might want to focus on something like salt tolerance, a trait predicted to be achieved through multiple pathways.  Then we can get an even better feel for how many options a population has for adaptation.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: nystatin, evolution, Saccharomyces cerevisiae, ergosterol biosynthesis

What Happens in Genes, Stays in Genes

September 06, 2012

Chromatin proteins, primarily histones, are a great way to control what parts of a cell’s DNA are accessible to its machinery.  These proteins coat the DNA and are marked up in certain ways to indicate how available a piece of DNA should be.  A methyl group here, an acetyl group there and a cell “knows” where the genes are that it is supposed to read!

Why doesn't RNA polymerase get a strike every time?

Of course this structure needs to be maintained or a cell might start to misread parts of its DNA as starting points of genes.  Then RNA polymerase II (RNAPII), the enzyme responsible for reading most protein-coding genes, would start making RNA from the wrong parts of the DNA, wreaking havoc in a cell.

One place where maintaining chromatin structure might be especially tricky is within the coding parts of genes.  It is easy to imagine RNAPII barreling down the DNA, knocking the proteins aside like pins in a bowling alley.  But it doesn’t.  For the most part the chromatin structure stays the same and survives the onslaught of an elongating RNAPII.

Two key marks for keeping histones in place are the trimethylation of lysine 36 of histone H3 (H3K36me3) that is mediated by Set2p, and a general deacetylation of histone H4 that is mediated by the Rpd3S histone deacetylase complex.  We know this because loss of either complex causes an increase in H4 acetylation and transcription starts from within genes.

In a recent study in Nature Structural & Molecular Biology, Smolle and coworkers identified two key components that help chromatin resist an elongating RNAPII in the yeast S. cerevisiae.  The first, called the Isw1b complex, binds H3K36me3 and the second, the Chd1 protein, binds RNAPII itself.  That these two were involved wasn’t surprising since previous work had suggested they helped prevent histone exchange at certain genes.

What makes this work unique is that the researchers showed the global importance of these proteins in the process and were able to tease out some of the fine details of what is going on at the molecular level. They used electrophoretic mobility shift assays to show that Isw1b bound the trimethylated form of H3 via its Ioc4p subunit and used chromosome immunoprecipitation coupled to microarrays (ChIP-chip) to show that Isw1b localized to the middle of genes in vivo. They also showed that when Set2p was removed, the localization disappeared (presumably because of the loss of the trimethylation of lysine 36).  They clearly demonstrated that Isw1b is found primarily in the middle of genes.

While these results indicate that the Ioc4p-containing Isw1b complex is moored to the middle of genes via its interaction with H3K36me3, it does not establish what it is doing there.  For this the researchers knocked out Isw1b and Chd1 and showed via genome tiling arrays a global increase in cryptic transcription starts.  The DNA in the middle of genes was now being used inappropriately by RNAPII as starting points for transcription.  Further investigation with Isw1b and Chd1 knockouts showed an increase in chromosome exchange and an increase in acetylated H4 in the middle of genes.

Whew.  So it appears that Isw1b and Chd1 inhibit inappropriate starts of transcription by keeping hypoacetylated histones in place over the parts of a gene that are read.   They are two of the key players in maintaining the right chromatin structure over genes.  They help keep RNAPII from railroading histones aside as it elongates, thus protecting the cell from inappropriate transcription starts.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: Chd1, chromatin, Isw1b, RNA polymerase II, Saccharomyces cerevisiae

Prions Let Yeast Take Traits for a Test Drive

August 27, 2012

For the most part, prions have a bad rep. They are the proverbial bad apple that spoils the whole bunch.

One bad apple can spoil the whole bunch...

A prion is a protein that misfolds in a certain way that creates a chain reaction to misfold many additional copies of that particular protein in a cell. This misfolding en masse can cause severe problems like mad cow disease or Alzheimer’s.

As if that weren’t bad enough, this misfoldedness can spread from one organism to another. Once a prion gets into a cell and/or a part of the body, it will cause many of its properly folded brethren to misfold too. This is true even though the prion gene in the new host is happily churning out properly folded protein.

These things look like a nightmare. Why on Earth are prions still around? Because in addition to their bad side, they can sometimes be an advantage too (at least in yeast).

In a study published in Nature in February 2012, Halfmann and coworkers provide compelling evidence that prions can help both laboratory and wild yeast strains to adapt rapidly to a changing environment, by unlocking survival traits hidden in yeast DNA. In other words, prions are a way for a yeast population to hedge its bets against a world of changing environments.

The authors focused on the most famous prion in yeast, the translation termination protein Sup35p. When Sup35p switches to prion mode ([PSI+]), it becomes bound up in insoluble fibers, causing translation termination to become leaky. Now normally untranslated parts of mRNAs become part of their respective proteins. And this can change these proteins’ functions.

Sure, most of this newfound variation will have no effect or maybe even be harmful, but occasionally the prion will reveal a beneficial trait. This yeast can then go on to survive and even thrive in this new environment.

This mechanism may apply to other prions in addition to Sup35p. Prions tend to come from proteins that are global regulators of transcription or translation. In the non-prion form, these proteins do their usual job making sure transcription and translation are following the rules. But when these proteins become misfolded into a prion, they can no longer perform their usual function. This uncovers previously silent bits of DNA or RNA for transcription or translation.

These authors also convincingly showed that prions are not some weird phenomenon found only in laboratory strains of yeast. They found evidence for prions in 255 out of the 690 wild strains they surveyed (although only ten had Sup35p based prions). Not only that, but many of these prions also conferred new traits on the yeast that could be beneficial in certain circumstances. It looks like prions may serve an important function in yeast.

A more surprising result from the study is that these prion-derived traits carry on in later generations even after the prion has been removed. For example, the authors looked at the wine yeast UCD978. They found that when Sup35p was in its prion form in this strain, UCD978 could effectively penetrate agar surfaces and that this trait was lost when the prion was cured, reverting Sup35p to its functional form.

They then took the study further and showed that after meiosis and sporulation, 5/30 haploid progeny of UCD978 retained the trait even after the prion was removed. These five had fixed the new trait and no longer required the prion to maintain it. They got all the benefits with none of the costs.

It isn’t obvious how this trait became independent of the original inducing prion. But that is for another study (or two or ten).

If the results of this study pan out, they show that prions are not just part of a disease but are really just another way to adapt to environmental changes and to pass them down to future generations. Maybe these apples aren’t so bad after all!

Prions allow yeast cells to take various traits out for a test drive.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: evolution, S. cerevisiae, prions, variation, SUP35

SGD Summer 2012 Newsletter

August 21, 2012

SGD sends out its quarterly newsletter to colleagues designated as contacts in SGD. This Summer 2012 newsletter is also available online. If you would like to receive this letter in the future please use the Colleague Submission/Update form to let us know.

Categories: Newsletter

GWAS Shows Potential in Yeast

August 09, 2012

Maybe GWAS will prove to be more useful in model organisms like yeast.

The idea behind a genome wide association study (GWAS) makes perfect sense.  Compare the DNA of one group of people with a disease to another group that doesn’t have the disease, identify the DNA region specific to the disease group, and then find the specific gene and mutations that lead to the disease.

In theory, this sort of study should have become routine once we had the human genome sequenced.  In practice, it has turned out to be less useful than everyone hoped.

Now, this doesn’t appear to be any fault with the technique itself.  Instead, it has more to do with the fact that many human diseases are simply too complex for GWAS to handle.

Most common human diseases appear to result from multiple genetic pathways and/or multiple genes.  Throw in environmental effects and GWAS quickly becomes overwhelmed.  At least for now, too many patients and controls would be needed for this powerful technique to have a real chance at deciphering most common human diseases.

But that doesn’t mean the technique isn’t useful.  It is very good at finding single genes involved in strongly expressed traits.  And this might be ideal for certain model organisms.

In a study just out in the latest issue of GENETICS, Connelly and Akey set out to investigate how well GWAS would work in the yeast, Saccharomyces cerevisiae.  In many respects, this yeast appears to be made for GWAS.

It has a small, easily sequenced genome, there is on average a polymorphism every 168 base pairs or so, and its linkage disequilibrium is low.  There are genome sequences from 36 wild and laboratory strains publicly available, all as diverse as can be. 

But this yeast isn’t perfect.  The chromosomal structure between strains tends to be much more varied than between two humans.  This is predicted to introduce a high error rate.  And this is just what Connelly and Akey saw when they ran some simulations.   

They found that the error rate was too high in the simulations to draw any meaningful conclusions.  But they also found that by using a more sophisticated analytical technique called EMMA, they were able to partly correct for some of these errors. 

Simulations are one thing, but how about real life?  Connelly and Akey next tested the method by applying it to a practical problem: identifying the genetic reasons for differences in mitochondrial DNA (mtDNA) copy number in yeast.  What they found mimicked the simulation data. 

Using more traditional analytical approaches on the data obtained from GWAS, they found 73 potential causative SNPs.  But when they switched to analyzing the data with EMMA, they found a single SNP that was significant.  It took a bit of hand waving, but the gene associated with this SNP could possibly be implicated in mtDNA copy number.  And then again, it might not.

This “significant” SNP was found amidst lots of errors and in a background of high p values.  In other words, this finding may not be a real one after all.  This experiment does not give confidence that GWAS can be used when all known strains of yeast are compared.

But if the strains to be included are selected more carefully, it may still prove to be a useful tool.  When Connelly and Akey focused on strains that were structurally similar, they found that the error rate was much lower.  Low enough that in the near term, scientists may be using GWAS to figure out how things work in model organisms.  

Hopefully the findings from GWAS applied to model organisms will illuminate disease mechanisms in humans. Then maybe GWAS can realize its full potential, although not in the way it was originally envisioned.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: GWAS, genome wide association study, yeast